The present invention relates to a probe configured to detect a polymorphism in an EGFR gene, an amplification primer, and the use thereof.
Epidermal growth factor receptor (EGFR) is the tyrosine kinase receptor for epidermal growth factor (EGF). EGFR is associated with a high frequency of expression in many solid tumors, and overexpression is known to be related to the malignancy or prognosis of a cancer. In this regard, gefitinib or the like that is an EGFR tyrosine kinase inhibitor (EGFR-TKI) is used as a therapeutic agent for cancers. However, a patient may exhibit an improvement in the effect of gefitinib in shrinking a tumor, or resistance to gefitinib may preclude a therapeutic effect. In recent years, the relationship between the sensitivity to this type of therapeutic agent and EGFR mutations has been elucidated. (PLoS Medicine, 2005. Vol. 2, No. 3, p. 225-235, and Journal of Clinical Oncology. 2005, Vol. 23, No. 11, p. 2513-2520).
These mutations are known to include a substitution mutation at position 790 and position 858 in EGFR, and a deletion mutation at exon 19 in the EGFR gene (PLoS Medicine, 2005, Vol. 2. No. 3, p. 225-235, and Journal of Clinical Oncology, 2005, Vol. 23. No. 1, p. 2513-2520). The mutation at position 790 is a mutation in which threonine (T) that is the amino acid at position 790 in EGFR is substituted by methionine (M). In the partial sequence of the EGFR gene illustrated in SEQ ID NO: 21, the cytosine (c) nucleotide at position 347 is substituted by thymin (t). The mutation at position 858 is a mutation in which leucine (L) that is the amino acid at position 858 in EGFR is substituted by arginine (R). In the partial sequence of the EGFR gene illustrated in SEQ ID NO: 1, the thymin (t) nucleotide at position 261 is substituted by guanine (g). The deletion mutation at exon 19 in the EGFR gene is a mutation in which several continuous nucleotides or more than ten continuous nucleotides are deleted in exon 19, and in the partial sequence of the EGFR gene illustrated in SEQ ID NO: 2, for example, any of the nucleotides at positions 112-164 are deleted. Therefore in the EGFR gene, there is the possibility of more effective and patient-specific cancer therapy if the presence or absence of a mutation (polymorphism) is detected to thereby evaluate sensitivity to gefitinib prior to therapy.
Various methods have been reported into relation to detection of polymorphisms in genes, and for example, include a method such as PCR restriction fragment length polymorphism (PCR-RFLP).
However, the PCR-RFLP method is labor intensive and in the event that an amplification product becomes dispersed, there is a risk of contamination of a separate subsequent reaction. These problems are related to difficulties associated with automatization of detection of polymorphisms.
In recent years, these problems have resulted in detection using melting curve analysis (Tm analysis) as a method of detection of gene polymorphism. In this method, a hybrid (double-stranded DNA) is formed from target single-stranded DNA from a detection sample and a probe that is complementary to the sequence of interest including the gene polymorphism that is the target of detection. Then a heating process is executed in relation to the hybridization product. The dissociation (melting) of the hybrid resulting from the temperature increase is detected by signal measurement using absorbance or the like to thereby determine the presence or absence of a target polymorphism by determining a Tm value based on the detection result. A Tm value increases corresponding to high sequence identity in the hybridization product, and decreases corresponding to low sequence identity. Consequently, a Tm value (evaluation reference value) can be obtained in advance in relation to the hybridization product of the sequence of interest including the gene polymorphism that is the target of detection and the complementary probe, and the Tm value (measurement value) of the target single-stranded DNA from a detection sample and the probe is measured. When the measurement value is the same as the evaluation reference value, it is determined that the target polymorphism is present in the target DNA. When the measurement value is lower than the evaluation reference value, it is determined that the target polymorphism is not present in the target DNA.
A melting curve may be obtained by performing the Tm analysis described herein. The melting curve may have a horizontal axis showing the temperature (° C.) during measurement, and a vertical axis showing the change in the fluorescent intensity.
“Tm value” is the temperature (dissociation temperature: Tm) at which double-stranded nucleic acid dissociates, and generally is defined as the temperature at which the absorbance at 260 nm reaches 50% of the total increase in absorbance. That is to say, when a solution containing double-stranded nucleic acid, for example double-stranded DNA, is heated, the absorbance at 260 nm increases. The increase results from the breakage of hydrogen bonds between the strands in the double-stranded DNA resulting from the heating and the dissociation into single-stranded DNA (DNA melting). When all double-stranded DNA dissociates to form single-stranded DNA, the absorbance exhibits approximately a 1.5 increase of the absorbance when commencing heating (absorbance when only double-stranded DNA is present). In this manner, completion of melting can be determined. The Tm value is set based on this phenomenon.
However, a detection method using this type of Tm analysis determines at least a one-nucleotide difference on the basis of the Tm value, and therefore when there is a plurality of genetic polymorphisms, the analysis of a single sample is extremely labor-intensive.